Sequence-determined dna fragments encoding trehalose-6p phosphatase proteins

ABSTRACT

The present invention provides DNA molecules that constitute fragments of the genome of a plant, and polypeptides encoded thereby. The DNA molecules are useful for specifying a gene product in cells, either as a promoter or as a protein coding sequence or as an UTR or as a 3′ termination sequence, and are also useful in controlling the behavior of a gene in the chromosome, in controlling the expression of a gene or as tools for genetic mapping, recognizing or isolating identical or related DNA fragments, or identification of a particular individual organism, or for clustering of a group of organisms with a common trait.

RELATED-APPLICATIONS

This application is (a) a continuation-in-part of U.S. patentapplication Ser. No. 13/718,219, filed Dec. 18, 2012, which is acontinuation-in-part of U.S. patent application Ser. No. 13/608,968,filed Sep. 10, 2012 (now U.S. Pat. No. 8,710,201), which is acontinuation-in-part of U.S. patent application Ser. No. 13/157,066,filed Jun. 9, 2011 (now U.S. Pat. No. 8,299,231), which is acontinuation-in-part-of U.S. patent application Ser. No. 12/639,295,filed Dec. 16, 2009 (now U.S. Pat. No. 7,989,609), which is acontinuation-in-part-of U.S. patent application Ser. No. 12/040,538,Filed Feb. 29, 2008 (now U.S. Pat. No. 7,659,386), which is acontinuation-in-part-of U.S. patent application Ser. No. 11/368,321,filed Mar. 3, 2006 (now U.S. Pat. No. 7,365,183), which is acontinuation-in-part-of U.S. patent application Ser. No. 11/006,231,filed Dec. 6, 2004, which is a continuation of U.S. patent applicationSer. No. 10/645,822 filed Aug. 22, 2003, and (b) a continuation-in-partof U.S. patent application Ser. No. 11/006,231 filed Dec. 6, 2004, whichis a continuation of U.S. patent application Ser. No. 10/645,822 filedAug. 22, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/462,849, filed Jun. 17, 2003, which claimspriority to U.S. Provisional Patent Application No. 60/389,140, filedJun. 17, 2002. The entire contents of these related applications areincorporated by reference in their entirety.

BACKGROUND

1. Technical Field

The present invention relates to isolated polynucleotides from plantsthat include a complete coding sequence, or a fragment thereof, that isexpressed. In addition, the present invention relates to polypeptides orproteins encoded by the coding sequence of these polynucleotides. Thepresent invention also relates to isolated polynucleotides thatrepresent regulatory regions of genes. The present invention alsorelates to isolated polynucleotides that represent untranslated regionsof genes. The present invention further relates to the use of theseisolated polynucleotides and polypeptides and proteins.

2. Background Information

There are more than 300,000 species of plants. They show a widediversity of forms, ranging from delicate liverworts, adapted for lifein a damp habitat, to cacti, capable of surviving in the desert. Theplant kingdom includes herbaceous plants, such as corn, whose life cycleis measured in months, to the giant redwood tree, which can live forthousands of years. This diversity reflects the adaptations of plants tosurvive in a wide range of habitats. This is seen most clearly in theflowering plants (phylum Angiospermophyta), which are the most numerous,with over 250,000 species. They are also the most widespread, beingfound from the tropics to the arctic.

When the molecular and genetic basis for different plant characteristicsare understood, a wide variety of polynucleotides, both endogenouspolynucleotides and created variants, polypeptides, cells, and wholeorganisms, can be exploited to engineer old and new plant traits in avast range of organisms including plants. These traits can range fromthe observable morphological characteristics, through adaptation tospecific environments to biochemical composition and to molecules thatthe plants (organisms) exude. Such engineering can involve tailoringexisting traits, such as increasing the production of taxol in yewtrees, to combining traits from two different plants into a singleorganism, such as inserting the drought tolerance of a cactus into acorn plant. Molecular and genetic knowledge also allows the creation ofnew traits. For example, the production of chemicals and pharmaceuticalsthat are not native to particular species or the plant kingdom as awhole.

SUMMARY

The present invention comprises polynucleotides, such as complete cDNAsequences and/or sequences of genomic DNA encompassing complete genes,fragments of genes, and/or regulatory elements of genes and/or regionswith other functions and/or intergenic regions, hereinafter collectivelyreferred to as Sequence-Determined DNA Fragments (SDFs) or sometimescollectively referred to as “genes or gene components”, or sometimes as“genes, gene components or products”, from different plant species,particularly corn, wheat, soybean, rice and Arabidopsis thaliana, andother plants and mutants, variants, fragments or fusions of said SDFsand polypeptides or proteins derived therefrom. In some instances, theSDFs span the entirety of a protein-coding segment. In some instances,the entirety of an mRNA is represented. Other objects of the inventionthat are also represented by SDFs of the invention are controlsequences, such as, but not limited to, promoters. Complements of anysequence of the invention are also considered part of the invention.

Other objects of the invention are polynucleotides comprising exonsequences, polynucleotides comprising intron sequences, polynucleotidescomprising introns together with exons, intron/exon junction sequences,5′ untranslated sequences, and 3′ untranslated sequences of the SDFs ofthe present invention. Polynucleotides representing the joinder of anyexons described herein, in any arrangement, for example, to produce asequence encoding any desirable amino acid sequence are within the scopeof the invention.

The present invention also resides in probes useful for isolating andidentifying nucleic acids that hybridize to an SDF of the invention. Theprobes can be of any length, but typically are 12-2000 nucleotides inlength; more typically, 15 to 200 nucleotides long; even more typically,18 to 100 nucleotides long.

Yet another object of the invention is a method of isolating and/oridentifying nucleic acids using the following steps: (a) contacting aprobe of the instant invention with a polynucleotide sample underconditions that permit hybridization and formation of a polynucleotideduplex; and (b) detecting and/or isolating the duplex of step (a).

The conditions for hybridization can be from low to moderate to highstringency conditions. The sample can include a polynucleotide having asequence unique in a plant genome. Probes and methods of the inventionare useful, for example, without limitation, for mapping of genetictraits and/or for positional cloning of a desired fragment of genomicDNA.

Probes and methods of the invention can also be used for detectingalternatively spliced messages within a species. Probes and methods ofthe invention can further be used to detect or isolate related genes inother plant species using genomic DNA (gDNA) and/or cDNA libraries. Insome instances, especially when longer probes and low to moderatestringency hybridization conditions are used, the probe will hybridizeto a plurality of cDNA and/or gDNA sequences of a plant. This approachis useful for isolating representatives of gene families which areidentifiable by possession of a common functional domain in the geneproduct or which have common cis-acting regulatory sequences. Thisapproach is also useful for identifying orthologous genes from otherorganisms.

The present invention also resides in constructs for modulating theexpression of the genes comprised of all or a fragment of an SDF. Theconstructs comprise all or a fragment of the expressed SDF, or of acomplementary sequence. Examples of constructs include ribozymescomprising RNA encoded by an SDF or by a sequence complementary thereto,antisense constructs, constructs comprising coding regions or partsthereof, constructs comprising promoters, introns, untranslated regions,scaffold attachment regions, methylating regions, enhancing or reducingregions, DNA and chromatin conformation modifying sequences, etc. Suchconstructs can be constructed using viral, plasmid, bacterial artificialchromosomes (BACs), plasmid artificial chromosomes (PACs), autonomousplant plasmids, plant artificial chromosomes or other types of vectorsand exist in the plant as autonomous replicating sequences or as DNAintegrated into the genome. When inserted into a host cell, theconstruct is, preferably, functionally integrated with, or operativelylinked to, a heterologous polynucleotide. For instance, a coding regionfrom an SDF might be operably linked to a promoter that is functional ina plant.

The present invention also resides in host cells, including bacterial oryeast cells or plant cells, and plants that harbor constructs such asdescribed above. Another aspect of the invention relates to methods formodulating expression of specific genes in plants by expression of thecoding sequence of the constructs, by regulation of expression of one ormore endogenous genes in a plant or by suppression of expression of thepolynucleotides of the invention in a plant. Methods of modulation ofgene expression include, without limitation, (1) inserting into a hostcell additional copies of a polynucleotide comprising a coding sequence;(2) modulating an endogenous promoter in a host cell; (3) insertingantisense or ribozyme constructs into a host cell; and (4) insertinginto a host cell a polynucleotide comprising a sequence encoding avariant, fragment, or fusion of the native polypeptides of the instantinvention.

BRIEF DESCRIPTION OF THE TABLES

The SDFs of the instant invention are listed in Table 2; annotationsrelevant to the sequences shown in Table 2 are presented in Table 1.Each sequence corresponds to a clone number. Each clone numbercorresponds to at least one sequence in Table 2. Nucleotide sequences inTable 2 are “Maximum Length Sequences” (MLS) that are the sequence of aninsert in a single clone.

Table 1 is a Reference Table which correlates each of the sequences andSEQ ID NOs in Table 2 with a corresponding Ceres clone number, Ceressequence identifier, and other information about the individualsequence. Table 2 is a Sequence Table with the sequence of each nucleicacid and amino acid sequence.

In Table 1, each section begins with a line that identifies thecorresponding internal Ceres clone by its ID number. Subsection (A) thenprovides information about the nucleotide sequence including thecorresponding sequence in Table 2, and the internal Ceres sequenceidentifier (“Ceres seq_id”). Subsection (B) provides similar informationabout a polypeptide sequence, but additionally identifies the locationof the start codon in the nucleotide sequence which codes for thepolypeptide. Subsection (C) provides information (where present)regarding identified domains within the polypeptide and (where present)a name for the polypeptide. Finally, subsection (D) provides (wherepresent) information concerning amino acids which are found to berelated and have some sequence identity to the polypeptide sequences ofTable 2. Those “related” sequences identified by a “gi” number are inthe GenBank data base.

In Table 2, Xaa within an amino acid sequence denotes an ambiguous aminoacid. An Xaa at the end of an amino acid sequence indicates a stopcodon.

TABLE 1 Reference table. (A) cDNA SEQ Pat. Appln. SEQ ID NO: 1 (SEQ IDNO: 57758 in U.S. Provisional Patent Application No. 60/389,140) CeresSEQ ID NO: 6782181 (B) PolyP SEQ Patent Appln. SEQ ID NO: 2 (SEQ ID NO:57759 in U.S. Provisional Patent Application No. 60/389,140) Ceres SEQID NO: 6782182 Loc. SEQ ID NO 1: @ 1 nt. (C) Pred. PP Norn. & Annot.(Dp) Rel. AA SEQ Align. NO 282145 gi No 19115342 Desp.: (NC 003424)trehalose-6p phosphatase [Schizosaccharomycespombe] >gi|7493523|pir||T38002 trehalose-6p phosphatase - fission yeast(Schizosaccharomyces pombe) >gi|11281551|pir||T43659 trehalose-6pphosphatase [Schizosaccharomyces pombe] % Idnt.: 28.1 Align. Len.: 221Loc. SEQ ID NO 2: 108 −> 309 aa.

TABLE 2 Sequence listing. <210> 1 <211> 1059 <212> DNA (genomic) <213>Oryza sativa subsp. indica <220> <221> misc_feature <222>(1) . . . (1059) <223> Ceres Seq. ID no. 6782181 <220> <221>misc_feature <222> ( ) . . . ( ) <223>n is a, c, t, g, unknown, or other <400> 1atggatttga gcaatagctc acctgtcatc accgatccgg tggcgatcag ccagcagttg   60ttgggcggcc tgccttcaaa tctgatgcag ttttcagtca tgcccggtgg ctactccagc  120tctggcatga acgttggtgt cagtaggctc aaaatcgagg aagtccttgt caatggactg  180cttgatgcca tgaaatcctc gtcacctcgc aggaggctga atgtagcatt tggcgaggac  240aattcatctg aagaagaaga ccctgcttac agcgcttgga tggcaaaatg tccttctgct  300ttggcttcct tcaagcaaat tgtagccagt gcacaaggga agaagattgc tgtgtttcta  360gactatgacg gcacactgtc gcctattgtg gatgatcctg acaaagcagt gatgtctccc  420gtgatgagag ctgctgtgag aaatgttgcg aagtacttcc ccactgcaat tgtcagcgga  480aggtcccgca ataaggtgtt tgaatttgta aaactgaagg agctttatta tgctggaagc  540catggtatgg acataatggc accttcagca aatcatgagc acagtgctga aaaggttacc  600aagtccctct tgcaagttgt cagtggaatt gaaggtgcaa ctgttgagaa caacaaattc  660tgcgtttctg tacattatcg caacgttgca gagaaggatt ggaaactggt cgcgcggctc  720gtaaacgaag tgctggaggc ttttcctcgt ctcaaagtaa ccaatggacg aatggtttta  780gaggttcgtc cggtgatcga ctgggacaag ggaaaggctg tggagtttct gctccagtca  840ctcgggctaa atgactctga aaatgtgatc cccatctaca ttggagacga cagaactgac  900gaagacgctt tcaaggtact tcgacagcga aattgcggtt atggaatact agtttcacag  960gttcccaagg aaactgaagc cttctactcg ctgagagatc catctgaagt gatggagttc 1020ctcaatttct tggtgagatg gaagaagcac tcagtgtga 1059 <210> 2 <211> 352 <212>PRT <213> Oryza sativa subsp. indica <220> <221> peptide <222>(1) . . . (352) <223> Ceres Seq. ID no. 6782182 <220> <221> misc_feature<222> ( ) . . . ( ) <223> xaa is any aa, unknown or other <400> 2Met Asp Leu Ser Asn Ser Ser Pro Val Ile Thr Asp Pro Val Ala Ile1               5                   10                  15Ser Gln Gln Leu Leu Gly Gly Leu Pro Ser Asn Leu Met Gln Phe Ser            20                  25                  30Val Met Pro Gly Gly Tyr Ser Ser Ser Gly Met Asn Val Gly Val Ser        35                  40                  45Arg Leu Lys Ile Glu Glu Val Leu Val Asn Gly Leu Leu Asp Ala Met    50                  55                  60Lys Ser Ser Ser Pro Arg Arg Arg Leu Asn Val Ala Phe Gly Glu Asp65                  70                  75                  80Asn Ser Ser Glu Glu Glu Asp Pro Ala Tyr Ser Ala Trp Met Ala Lys                85                  90                  95Cys Pro Ser Ala Leu Ala Ser Phe Lys Gln Ile Val Ala Ser Ala Gln            100                 105                 110Gly Lys Lys Ile Ala Val Phe Leu Asp Tyr Asp Gly Thr Leu Ser Pro        115                 120                 125Ile Val Asp Asp Pro Asp Lys Ala Val Met Ser Pro Val Met Arg Ala    130                 135                 140Ala Val Arg Asn Val Ala Lys Tyr Phe Pro Thr Ala Ile Val Ser Gly145                 150                 155                 160Arg Ser Arg Asn Lys Val Phe Glu Phe Val Lys Leu Lys Glu Leu Tyr                165                 170                 175Tyr Ala Gly Ser His Gly Met Asp Ile Met Ala Pro Ser Ala Asn His            180                 185                 190Glu His Ser Ala Glu Lys Val Thr Lys Ser Leu Leu Gln Val Val Ser        195                 200                 205Gly Ile Glu Gly Ala Thr Val Glu Asn Asn Lys Phe Cys Val Ser Val    210                 215                 220His Tyr Arg Asn Val Ala Glu Lys Asp Trp Lys Leu Val Ala Arg Leu225                 230                 235                 240Val Asn Glu Val Leu Glu Ala Phe Pro Arg Leu Lys Val Thr Asn Gly                245                 250                 255Arg Met Val Leu Glu Val Arg Pro Val Ile Asp Trp Asp Lys Gly Lys            260                 265                 270Ala Val Glu Phe Leu Leu Gln Ser Leu Gly Leu Asn Asp Ser Glu Asn        275                 280                 285Val Ile Pro Ile Tyr Ile Gly Asp Asp Arg Thr Asp Glu Asp Ala Phe    290                 295                 300Lys Val Leu Arg Gln Arg Asn Cys Gly Tyr Gly Ile Leu Val Ser Gln305                 310                 315                 320Val Pro Lys Glu Thr Glu Ala Phe Tyr Ser Leu Arg Asp Pro Ser Glu                325                 330                 335Val Met Glu Phe Leu Asn Phe Leu Val Arg Trp Lys Lys His Ser Val            340                 345                 350

DETAILED DESCRIPTION

The invention relates to polynucleotides and methods of use thereof,such as probes, primers and substrates; methods of detection andisolation; hybridization; methods of mapping; southern blotting;isolating cDNA from related organisms; isolating and/or identifyingorthologous genes; methods of inhibiting gene expression (e.g.,antisense, ribozyme constructs, chimeraplasts, co-suppression,transcriptional silencing, and other methods to inhibit geneexpression); methods of functional analysis; promoter sequences andtheir use; utrs and/or intron sequences and their use; and codingsequences and their use.

The invention also relates to polypeptides and proteins and methods ofuse thereof, such as native polypeptides and proteins; antibodies; invitro applications; polypeptide variants, fragments and fusions.

The invention also includes methods of modulating polypeptideproduction, such as suppression (e.g., antisense, ribozymes,co-suppression, insertion of sequences into the gene to be modulated,promoter modulation, expression of genes containing dominant-negativemutations) and enhanced expression (e.g., insertion of an exogenous geneand promoter modulation).

The invention further concerns gene constructs and vector construction,such as coding sequences, promoters, and signal peptides. the inventionstill further relates to transformation techniques.

Polynucleotides

Exemplified SDFs of the invention represent fragments of the genome ofcorn, wheat, rice, soybean or Arabidopsis and/or represent mRNAexpressed from that genome. The isolated nucleic acid of the inventionalso encompasses corresponding fragments of the genome and/or cDNAcomplement of other organisms as described in detail below.

Polynucleotides of the invention can be isolated from polynucleotidelibraries using primers comprising sequences similar to those describedin the attached Table 2 or complements thereof. See, for example, themethods described in Sambrook et al. (Molecular Cloning, a LaboratoryManual, 2nd ed., c. 1989 by Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Alternatively, the polynucleotides of the invention can be produced bychemical synthesis. Such synthesis methods are described below.

It is contemplated that the nucleotide sequences presented herein maycontain some small percentage of errors. These errors may arise in thenormal course of determination of nucleotide sequences. Sequence errorscan be corrected by obtaining seeds such as those deposited under theaccession numbers cited herein, propagating them, isolating genomic DNAor appropriate mRNA from the resulting plants or seeds thereof,amplifying the relevant fragment of the genomic DNA or mRNA usingprimers having a sequence that flanks the erroneous sequence, andsequencing the amplification product.

Probes, Primers and Substrates

Probes and primers of the instant invention will hybridize to apolynucleotide comprising a sequence in Table 2. Though many differentnucleotide sequences can encode an amino acid sequence, in someinstances, the sequences of Table 2 are preferred for encodingpolypeptides of the invention. However, the sequence of the probesand/or primers of the instant invention need not be identical to thosein Table 2 or the complements thereof. Some variation in the sequenceand length can lead to increase assay sensitivity if the nucleic acidprobe can form a duplex with a target nucleotide in a sample that can bedetected or isolated. The probes and/or primers of the invention caninclude additional nucleotides that may be helpful as a label to detectthe formed duplex or for later cloning purposes.

Probe length will vary depending on the application. For use as a PCRprimer, probes should be 12-40 nucleotides, preferably 18-30 nucleotideslong. For use in mapping, probes should be 50 to 500 nucleotides,preferably 100-250 nucleotides long. For Southern hybridizations, probesas long as several kilobases can be used as explained below.

The probes and/or primers can be produced by synthetic procedures suchas the triester method of Matteucci et al. (J. Am. Chem. Soc., 103:3185(1981)); or according to Urdea et al. (Proc. Natl. Acad. Sci. USA,80:7461 (1981)) or using commercially available automatedoligonucleotide synthesizers.

Methods of Detection and Isolation

The polynucleotides of the invention can be utilized in a number ofmethods known to those skilled in the art as probes and/or primers toisolate and detect polynucleotides, including, without limitation:Southern blot assays, Northern blot assays, Branched DNA hybridizationassays, polymerase chain reaction, and microarray assays, and variationsthereof. Specific methods given by way of examples, and discussed belowinclude: hybridization, methods of mapping, Southern blotting, isolatingcDNA from related organisms, and isolating and/or identifyingorthologous genes.

Hybridization

The isolated SDFs of Tables 1 and 2 can be used as probes and/or primersfor detection and/or isolation of related polynucleotide sequencesthrough hybridization. Hybridization of one nucleic acid to anotherconstitutes a physical property that defines the subject SDF of theinvention and the identified related sequences. Also, such hybridizationimposes structural limitations on the pair. A good general discussion ofthe factors for determining hybridization conditions is provided bySambrook et al. (Molecular Cloning, a Laboratory Manual, 2nd ed., c.1989 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;see esp., chapters 11 and 12). Additional considerations and details ofthe physical chemistry of hybridization are provided by Keller and Manak(DNA Probes, 2^(nd) Ed. pp. 1-25, c. 1993 by Stockton Press, New York,N.Y.).

Depending on the stringency of the conditions under which these probesand/or primers are used, polynucleotides exhibiting a wide range ofsimilarity to those in Tables 1 or 2 or fragments thereof can bedetected or isolated. When the practitioner wishes to examine the resultof membrane hybridizations under a variety of stringencies, an efficientway to do so is to perform the hybridization under a low stringencycondition, then to wash the hybridization membrane under increasinglystringent conditions.

When using SDFs to identify orthologous genes in other species, thepractitioner will preferably adjust the amount of target DNA of eachspecies so that, as nearly as is practical, the same number of genomeequivalents are present for each species examined. This prevents faintsignals from species having large genomes, and thus small numbers ofgenome equivalents per mass of DNA, from erroneously being interpretedas absence of the corresponding gene in the genome.

The probes and/or primers of the instant invention can also be used todetect or isolate nucleotides that are “identical” to the probes orprimers. Two nucleic acid sequences or polypeptides are said to be“identical” if the sequence of nucleotides or amino acid residues,respectively, in the two sequences is the same when aligned for maximumcorrespondence as described below.

Isolated polynucleotides within the scope of the invention also includeallelic variants of the specific sequences presented in Tables 1 and 2.The probes and/or primers of the invention can also be used to detectand/or isolate polynucleotides exhibiting at least 80% sequence identitywith the sequences of Table 1 or 2.

With respect to nucleotide sequences, degeneracy of the genetic codeprovides the possibility to substitute at least one base of the basesequence of a gene with a different base without causing the amino acidsequence of the polypeptide produced from the gene to be changed. Hence,the DNA of the present invention may also have any base sequence thathas been changed from a sequence in Table 1 or 2 by substitution inaccordance with degeneracy of genetic code. References describing codonusage include: Carels et al., (J. Mol. Evol., 46:45 (1998)) and Fennoyet al. (Nucl. Acids Res., 21(23):5294 (1993)).

Mapping

The isolated SDFs provided herein can be used to create various types ofgenetic and physical maps of the genome of corn, Arabidopsis, soybean,rice, wheat, or other plants. Some SDFs may be absolutely associatedwith particular phenotypic traits, allowing construction of grossgenetic maps. While not all SDFs of Table 2 of the priority patentapplications will immediately be associated with a phenotype, all SDFscan be used as probes for identifying polymorphisms associated withphenotypes of interest. Briefly, one method of mapping involves totalDNA isolation from individuals. It is subsequently cleaved with one ormore restriction enzymes, separated according to mass, transferred to asolid support, hybridized with SDF DNA, and the pattern of fragmentscompared. Polymorphisms associated with a particular SDF are visualizedas differences in the size of fragments produced between individual DNAsamples after digestion with a particular restriction enzyme andhybridization with the SDF. After identification of polymorphic SDFsequences, linkage studies can be conducted. By using the individualsshowing polymorphisms as parents in crossing programs, F2 progenyrecombinants or recombinant inbreds, for example, are then analyzed. Theorder of DNA polymorphisms along the chromosomes can be determined basedon the frequency with which they are inherited together versusindependently. The closer two polymorphisms are together in achromosome; the higher the probability that they are inherited together.Integration of the relative positions of all the polymorphisms andassociated marker SDFs can produce a genetic map of the species, wherethe distances between markers reflect the recombination frequencies inthat chromosome segment.

The use of recombinant inbred lines for such genetic mapping isdescribed for Arabidopsis by Alonso-Blanco et al. (Methods in MolecularBiology, vol. 82, “Arabidopsis Protocols”, pp. 137-146, J. M.Martinez-Zapater and J. Salinas, eds., c. 1998 by Humana Press, Totowa,N.J.) and for corn by Burr (“Mapping Genes with Recombinant Inbreds”,pp. 249-254. In Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c.1994 by Springer-Verlag New York, Inc.: New York, N.Y., USA; BerlinGermany; Burr et al., Genetics, 118:519 (1998); and Gardiner et al.,Genetics, 134:917 (1993)). This procedure, however, is not limited toplants and can be used for other organisms such as yeast or forindividual cells.

The SDFs provided herein can also be used for simple sequence repeat(SSR) mapping. Rice SSR mapping is described elsewhere (Morgante et al.,The Plant Journal, 3:165 (1993)), Panaud et al., Genome, 38:1170 (1995);Senior et al., Crop Science, 36:1676 (1996), Taramino et al., Genome,39:277 (1996); and Ahn et al., Molecular and General Genetics,241:483-90 (1993)). SSR mapping can be achieved using various methods.In one instance, polymorphisms are identified when sequence specificprobes contained within an SDF flanking an SSR are made and used inpolymerase chain reaction (PCR) assays with template DNA from two ormore individuals of interest. Here, a change in the number of tandemrepeats between the SSR-flanking sequences produces differently sizedfragments (U.S. Pat. No. 5,766,847). Alternatively, polymorphisms can beidentified by using the PCR fragment produced from the SSR-flankingsequence specific primer reaction as a probe against Southern blotsrepresenting different individuals (Refseth et al., Electrophoresis,18:1519 (1997)).

Genetic and physical maps of crop species have many uses. For example,these maps can be used to devise positional cloning strategies forisolating novel genes from the mapped crop species. In addition, becausethe genomes of closely related species are largely syntenic (that is,they display the same ordering of genes within the genome), these mapscan be used to isolate novel alleles from relatives of crop species bypositional cloning strategies.

The various types of maps discussed above can be used with the SDFsprovided herein to identify Quantitative Trait Loci (QTLs). Manyimportant crop traits, such as the solids content of tomatoes, arequantitative traits and result from the combined interactions of severalgenes. These genes reside at different loci in the genome, oftentimes ondifferent chromosomes, and generally exhibit multiple alleles at eachlocus. The SDFs provided herein can be used to identify QTLs and isolatespecific alleles as described by de Vicente and Tanksley (Genetics134:585 (1993)). In addition to isolating QTL alleles in present cropspecies, the SDFs provided herein can also be used to isolate allelesfrom the corresponding QTL of wild relatives. Transgenic plants havingvarious combinations of QTL alleles can then be created, and the effectsof the combinations measured. Once a desired allele combination has beenidentified, crop improvement can be accomplished either throughbiotechnological means or by directed conventional breeding programs(for review, see Tanksley and McCouch, Science, 277:1063 (1997)).

In another embodiment, the SDFs provided herein can be used to helpcreate physical maps of the genome of corn, Arabidopsis, and relatedspecies. Where SDFs have been ordered on a genetic map, as describedabove, they can be used as probes to discover which clones in largelibraries of plant DNA fragments in YACs, BACs, etc. contain the sameSDF or similar sequences, thereby facilitating the assignment of thelarge DNA fragments to chromosomal positions. Subsequently, the largeBACs, YACs, etc. can be ordered unambiguously by more detailed studiesof their sequence composition (see, e.g., Marra et al., GenomicResearch, 7:1072-1084 (1997)) and by using their end or other sequencesto find the identical sequences in other cloned DNA fragments. Theoverlapping of DNA sequences in this way allows large contigs of plantsequences to be built that, when sufficiently extended, provide acomplete physical map of a chromosome. Sometimes the SDFs themselveswill provide the means of joining cloned sequences into a contig.

The patent publication WO95/35505 and U.S. Pat. Nos. 5,445,943 and5,410,270 describe scanning multiple alleles of a plurality of lociusing hybridization to arrays of oligonucleotides. These techniques areuseful for each of the types of mapping discussed above.

Following the procedures described above and using a plurality of theSDFs of Table 2 or Table 2 on any of the priority patent applications,any individual can be genotyped. These individual genotypes can be usedfor the identification of particular cultivars, varieties, lines,ecotypes, and genetically modified plants or can serve as tools forsubsequent genetic studies involving multiple phenotypic traits.

Southern Blot Hybridization

The sequences of Tables 1 and 2 can be used as probes for varioushybridization techniques. These techniques are useful for detectingtarget polynucleotides in a sample or for determining whether transgenicplants, seeds or host cells harbor a gene or sequence of interest andthus might be expected to exhibit a particular trait or phenotype.

In addition, the SDFs provided herein can be used to isolate additionalmembers of gene families from the same or different species and/ororthologous genes from the same or different species. This isaccomplished by hybridizing an SDF to, for example, a Southern blotcontaining the appropriate genomic DNA or cDNA. Given the resultinghybridization data, one of ordinary skill in the art could distinguishand isolate the correct DNA fragments by size, restriction sites,sequence, and stated hybridization conditions from a gel or from alibrary.

Identification and isolation of orthologous genes from closely relatedspecies and alleles within a species is particularly desirable becauseof their potential for crop improvement. Many important crop traits,such as the solid content of tomatoes, result from the combinedinteractions of the products of several genes residing at different lociin the genome. Generally, alleles at each of these loci can makequantitative differences to the trait. By identifying and isolatingnumerous alleles for each locus from within or different species,transgenic plants with various combinations of alleles can be created,and the effects of the combinations measured. Once a more favorableallele combination has been identified, crop improvement can beaccomplished either through biotechnological means or by directedconventional breeding programs (Tanksley et al., Science, 277:1063(1997)).

The results from hybridizations of an SDFs provided herein to, forexample, Southern blots containing DNA from another species can also beused to generate restriction fragment maps for the corresponding genomicregions. These maps provide additional information about the relativepositions of restriction sites within fragments, further distinguishingmapped DNA from the remainder of the genome. Physical maps can be madeby digesting genomic DNA with different combinations of restrictionenzymes.

Probes for Southern blotting to distinguish individual restrictionfragments can range in size from 15 to 20 nucleotides to severalthousand nucleotides. More preferably, the probe is 100 to 1,000nucleotides long for identifying members of a gene family when it isfound that repetitive sequences would complicate the hybridization. Foridentifying an entire corresponding gene in another species, the probeis more preferably the length of the gene, typically 2,000 to 10,000nucleotides, but probes 50-1,000 nucleotides long might be used. Somegenes, however, might require probes up to 1,500 nucleotides long oroverlapping probes constituting the full-length sequence to span theirlengths.

Also, while it is preferred that the probe be homogeneous with respectto its sequence, it is not necessary. For example, as described below, aprobe representing members of a gene family having diverse sequences canbe generated using PCR to amplify genomic DNA or RNA templates usingprimers derived from SDFs that include sequences that define the genefamily.

For identifying corresponding genes in another species, the next mostpreferable probe is a cDNA spanning the entire coding sequence, whichallows all of the mRNA-coding fragment of the gene to be identified.Probes for Southern blotting can easily be generated from SDFs by makingprimers having the sequence at the ends of the SDF and using corn orArabidopsis genomic DNA as a template. In instances where the SDFincludes sequence conserved among species, primers including theconserved sequence can be used for PCR with genomic DNA from a speciesof interest to obtain a probe.

Similarly, if the SDF includes a domain of interest, that fragment ofthe SDF can be used to make primers and, with appropriate template DNA,used to make a probe to identify genes containing the domain.Alternatively, the PCR products can be resolved, for example by gelelectrophoresis, and cloned and/or sequenced. Using Southernhybridization, the variants of the domain among members of a genefamily, both within and across species, can be examined.

Isolating DNA from Related Organisms

The SDFs provided herein can be used to isolate the corresponding DNAfrom other organisms. Either cDNA or genomic DNA can be isolated. Forisolating genomic DNA, a lambda, cosmid, BAC, or YAC, or other largeinsert genomic library from the plant of interest can be constructedusing standard molecular biology techniques as described in detail bySambrook et al. (Molecular Cloning: A Laboratory Manual, 2^(nd) ed. ColdSpring Harbor Laboratory Press, New York (1989)) and by Ausubel et al.(Current Protocols in Molecular Biology, Greene Publishing, New York(1992)).

To screen a phage library, for example, recombinant lambda clones areplated out on appropriate bacterial medium using an appropriate E. colihost strain. The resulting plaques are lifted from the plates usingnylon or nitrocellulose filters. The plaque lifts are processed throughdenaturation, neutralization, and washing treatments following thestandard protocols outlined by Ausubel et al. (Current Protocols inMolecular Biology, Greene Publishing, New York (1992)). The plaque liftsare hybridized to either radioactively labeled or non-radioactivelylabeled SDF DNA at room temperature for about 16 hours, usually in thepresence of 50% formamide and 5×SSC (sodium chloride and sodium citrate)buffer and blocking reagents. The plaque lifts are then washed at 42° C.with 1% Sodium Dodecyl Sulfate (SDS) and at a particular concentrationof SSC. The SSC concentration used is dependent upon the stringency atwhich hybridization occurred in the initial Southern blot analysisperformed. For example, if a fragment hybridized under medium stringency(e.g., Tm-20° C.), then this condition is maintained or preferablyadjusted to a less stringent condition (e.g., Tm-30° C.) to wash theplaque lifts. Positive clones show detectable hybridization e.g., byexposure to X-ray films or chromogen formation. The positive clones arethen subsequently isolated for purification using the same generalprotocol outlined above. Once the clone is purified, restrictionanalysis can be conducted to narrow the region corresponding to the geneof interest. The restriction analysis and succeeding subcloning stepscan be done using procedures described by, for example, Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2^(nd) ed. Cold Spring HarborLaboratory Press, New York (1989)).

The procedures outlined for the lambda library are essentially similarto those used for YAC library screening, except that the YAC clones areharbored in bacterial colonies. The YAC clones are plated out atreasonable density on nitrocellulose or nylon filters supported byappropriate bacterial medium in petri plates. Following the growth ofthe bacterial clones, the filters are processed through thedenaturation, neutralization, and washing steps following the proceduresof Ausubel et al. (Current Protocols in Molecular Biology, GreenePublishing, New York (1992)). The same hybridization procedures forlambda library screening are followed.

To isolate cDNA, similar procedures using appropriately modified vectorsare employed. For instance, the library can be constructed in a lambdavector appropriate for cloning cDNA such as λgt11. Alternatively, thecDNA library can be made in a plasmid vector. cDNA for cloning can beprepared by any of the methods known in the art, but is preferablyprepared as described above. Preferably, a cDNA library will include ahigh proportion of full-length clones.

Isolating and/or Identifying Orthologous Genes

The probes and primers provided herein can be used to identify and/orisolate polynucleotides related to those set forth in Tables 1 and 2.Related polynucleotides are those that are native to other plantorganisms and exhibit either similar sequence or encode polypeptideswith similar biological activity. One specific example is an orthologousgene. Orthologous genes have the same functional activity. As such,orthologous genes may be distinguished from homologous genes. Thepercentage of identity is a function of evolutionary separation and, inclosely related species, the percentage of identity can be 98 to 100%.The amino acid sequence of a protein encoded by an orthologous gene canbe less than 75% identical, but tends to be at least 75% or at least 80%identical, more preferably at least 90%, most preferably at least 95%identical to the amino acid sequence of the reference protein.

To find orthologous genes, the probes are hybridized to nucleic acidsfrom a species of interest under low stringency conditions, preferablyone where sequences containing as much as 40-45% mismatches will be ableto hybridize. This condition is established by T_(m)-40° C. to Tm-48° C.(see below). Blots are then washed under conditions of increasingstringency. It is preferable that the wash stringency be such thatsequences that are 85 to 100% identical will hybridize. More preferably,sequences 90 to 100% identical will hybridize, and most preferably onlysequences greater than 95% identical will hybridize. One of ordinaryskill in the art will recognize that, due to degeneracy in the geneticcode, amino acid sequences that are identical can be encoded by DNAsequences as little as 67% identical or less. Thus, it is preferable,for example, to make an overlapping series of shorter probes, on theorder of 24 to 45 nucleotides, and individually hybridize them to thesame arrayed library to avoid the problem of degeneracy introducinglarge numbers of mismatches.

As evolutionary divergence increases, genome sequences also tend todiverge. Thus, one of skill will recognize that searches for orthologousgenes between more divergent species will require the use of lowerstringency conditions compared to searches between closely relatedspecies. Also, degeneracy of the genetic code is more of a problem forsearches in the genome of a species more distant evolutionarily from thespecies that is the source of the SDF probe sequences.

Therefore, the methods described by Bouckaert et al. (U.S. ProvisionalPatent Application Ser. No. 60/121,700; filed Feb. 25, 1999 and herebyincorporated in its entirety by reference) can be applied to the SDFsprovided herein to isolate related genes from plant species which do nothybridize to the corn Arabidopsis, soybean, rice, wheat, and other plantsequences provided in Tables 1 and 2.

Identification of the relationship of nucleotide or amino acid sequencesamong plant species can be done by comparing the nucleotide or aminoacid sequences of SDFs provided herein with nucleotide or amino acidsequences of other SDFs such as those provided in Table 2 of any of thepriority applications.

The SDFs provided herein can also be used as probes to search for genesthat are related to the SDF within a species. Such related genes aretypically considered to be members of a gene family. In such a case, thesequence similarity will often be concentrated into one or a fewfragments of the sequence. The fragments of similar sequence that definethe gene family typically encode a fragment of a protein or RNA that hasan enzymatic or structural function. The percentage of identity in theamino acid sequence of the domain that defines the gene family ispreferably at least 70%, more preferably 80 to 95%, most preferably 85to 99%. To search for members of a gene family within a species, a lowstringency hybridization is usually performed, but this will depend uponthe size, distribution and degree of sequence divergence of domains thatdefine the gene family. SDFs in Table 2 of any of the priority patentapplications that encompass regulatory regions can be used to identifycoordinately expressed genes by using the regulatory region sequence ofthe SDF as a probe.

In the instances where the SDFs are identified as being expressed fromgenes that confer a particular phenotype, then the SDFs can also be usedas probes to assay plants of different species for those phenotypes.

Methods to Inhibit Gene Expression

The nucleic acid molecules provided herein can be used to inhibit genetranscription and/or translation. Examples of such methods and materialsinclude, without limitation, antisense constructs, ribozyme constructs,chimeraplast constructs, co-suppression, transcriptional silencing, andother methods of gene expression.

Antisense

In some instances, it is desirable to suppress expression of anendogenous or exogenous gene. A well-known instance is the FLAVOR-SAVOR™tomato, in which the gene encoding ACC synthase is inactivated by anantisense approach, thus delaying softening of the fruit after ripening.See, for example, U.S. Pat. No. 5,859,330; U.S. Pat. No. 5,723,766;Oeller et al., Science, 254:437-439 (1991); and Hamilton et al., Nature,346:284-287 (1990). Also, timing of flowering can be controlled bysuppression of the FLOWERING LOCUS C (FLC). High levels of thistranscript are associated with late flowering, while absence of FLC isassociated with early flowering (Michaels et al., Plant Cell, 11:949(1999)). Also, the transition of apical meristem from production ofleaves with associated shoots to flowering is regulated by TERMINALFLOWER1, APETALA1 and LEAFY. Thus, when it is desired to induce atransition from shoot production to flowering, it is desirable tosuppress TFL1 expression (Liljegren, Plant Cell, 11:1007 (1999)). Asanother instance, arrested ovule development and female sterility resultfrom suppression of the ethylene forming enzyme but can be reversed byapplication of ethylene (De Martinis et al., Plant Cell, 11:1061(1999)). The ability to manipulate female fertility of plants is usefulin increasing fruit production and creating hybrids.

In the case of polynucleotides used to inhibit expression of anendogenous gene, the introduced sequence need not be perfectly identicalto a sequence of the target endogenous gene. The introducedpolynucleotide sequence will typically be at least substantiallyidentical to the target endogenous sequence.

Some polynucleotide SDFs provided herein or provided in Table 2 of anyof the priority patent applications represent sequences that areexpressed in corn, wheat, rice, soybean, Arabidopsis, and/or otherplants. Any of these sequences can be used to generate antisenseconstructs to inhibit translation and/or degradation of transcripts ofan SDFs, typically in a plant cell.

To accomplish this, a polynucleotide segment from the desired gene thatcan hybridize to the mRNA expressed from the desired gene (the“antisense segment”) is operably linked to a promoter such that theantisense strand of RNA will be transcribed when the construct ispresent in a host cell. A regulated promoter can be used in theconstruct to control transcription of the antisense segment so thattranscription occurs only under desired circumstances.

The antisense segment to be introduced generally will be substantiallyidentical to at least a fragment of the endogenous gene or genes to berepressed. The sequence, however, need not be perfectly identical toinhibit expression. Further, the antisense product may hybridize to theuntranslated region instead of or in addition to the coding sequence ofthe gene. The vectors provided herein can be designed such that theinhibitory effect applies to other proteins within a family of genesexhibiting homology or substantial homology to the target gene.

For antisense suppression, the introduced antisense segment sequencealso need not be full length relative to either the primarytranscription product or the fully processed mRNA. Generally, a higherpercentage of sequence identity can be used to compensate for the use ofa shorter sequence. Furthermore, the introduced sequence need not havethe same intron or exon pattern, and homology of non-coding segments maybe equally effective. Normally, a sequence of between about 30 or 40nucleotides and the full length of the transcript can be used, though asequence of at least about 100 nucleotides is preferred, a sequence ofat least about 200 nucleotides is more preferred, and a sequence of atleast about 500 nucleotides is especially preferred.

Chimeraplasts

The SDFs provided herein, such as those described in Table 2, can alsobe used to construct chimeraplasts that can be introduced into a cell toproduce at least one specific nucleotide change in a sequence. Achimeraplast is an oligonucleotide comprising DNA and/or RNA thatspecifically hybridizes to a target region in a manner which creates amismatched base-pair. This mismatched base-pair signals the cell'srepair enzyme machinery which acts on the mismatched region resulting inthe replacement, insertion, or deletion of designated nucleotide(s). Thealtered sequence is then expressed by the cell's normal cellularmechanisms. Chimeraplasts can be designed to repair mutant genes, modifygenes, introduce site-specific mutations, and/or act to interrupt oralter normal gene function. See, e.g., U.S. Pat. Nos. 6,010,907 and6,004,804 and PCT Publication Nos. WO99/58723 and WO99/07865.

Sense Suppression

The SDFs provided herein, such as those described in Table 2, are alsouseful to modulate gene expression by sense suppression. Sensesuppression represents another method of gene suppression by introducingat least one exogenous copy or fragment of the endogenous sequence to besuppressed.

Introduction of expression cassettes in which a nucleic acid isconfigured in the sense orientation with respect to the promoter intothe chromosome of a plant or by a self-replicating virus has been shownto be an effective means by which to induce degradation of mRNAs oftarget genes. An example of the use of this method to modulateexpression of endogenous genes is provided elsewhere (Napoli et al., ThePlant Cell, 2:279 (1990), and U.S. Pat. Nos. 5,034,323; 5,231,020; and5,283,184) Inhibition of expression may require some transcription ofthe introduced sequence.

For sense suppression, the introduced sequence generally will besubstantially identical to the endogenous sequence intended to beinactivated. The minimal percentage of sequence identity will typicallybe greater than about 65%, but a higher percentage of sequence identitymight exert a more effective reduction in the level of normal geneproducts. Sequence identity of more than about 80% is preferred, thoughabout 95% to absolute identity would be most preferred. As withantisense regulation, the effect would likely apply to any otherproteins within a similar family of genes exhibiting homology orsubstantial homology to the suppressing sequence.

Transcriptional Silencing

The nucleic acid sequences provided herein or provided in Table 2 of anyof the priority patent applications (and fragments thereof) containsequences that can be inserted into the genome of an organism resultingin transcriptional silencing. Such regulatory sequences need not beoperatively linked to coding sequences to modulate transcription of agene. Specifically, a promoter sequence without any other element of agene can be introduced into a genome to transcriptionally silence anendogenous gene (see, for example, Vaucheret et al., The Plant Journal,16:651-659 (1998)). As another example, triple helices can be formedusing oligonucleotides based on sequences from Table 2 provided hereinor Table 2 of any of the priority patent applications, fragmentsthereof, and substantially similar sequence thereto. The oligonucleotidecan be delivered to the host cell and can bind to the promoter in thegenome to form a triple helix and prevent transcription. Anoligonucleotide of interest is one that can bind to the promoter andblock binding of a transcription factor to the promoter. In such a case,the oligonucleotide can be complementary to the sequences of thepromoter that interact with transcription binding factors.

Other Methods to Inhibit Gene Expression

Yet another means of suppressing gene expression is to insert apolynucleotide into the gene of interest to disrupt transcription ortranslation of the gene.

Low frequency homologous recombination can be used to target apolynucleotide insert to a gene by flanking the polynucleotide insertwith sequences that are substantially similar to the gene to bedisrupted. Sequences from Table 2 provided herein or Table 2 of any ofthe priority patent applications, fragments thereof, and substantiallysimilar sequence thereto can be used for homologous recombination.

In addition, random insertion of polynucleotides into a host cell genomecan also be used to disrupt the gene of interest (Azpiroz-Leehan et al.,Trends in Genetics, 13:152 (1997)). In this method, screening for clonesfrom a library containing random insertions is preferred to identifyingthose that have polynucleotides inserted into the gene of interest. Suchscreening can be performed using probes and/or primers described abovebased on sequences from Table 2 provided herein or Table 2 of any of thepriority patent applications, fragments thereof, and substantiallysimilar sequence thereto. The screening can also be performed byselecting clones or R₁ plants having a desired phenotype.

Methods of Functional Analysis

The constructs described in the methods provided herein can be used todetermine the function of the polypeptide encoded by the gene that istargeted by the constructs.

Down-regulating the transcription and translation of the targeted genein the host cell or organisms, such as a plant, may produce phenotypicchanges as compared to a wild-type cell or organism. In addition, invitro assays can be used to determine if any biological activity, suchas calcium flux, DNA transcription, nucleotide incorporation, etc. arebeing modulated by the down-regulation of the targeted gene.

Coordinated regulation of sets of genes, e.g., those contributing to adesired polygenic trait, is sometimes necessary to obtain a desiredphenotype. SDFs provided in Table 2 or Table 2 of any of the prioritypatent applications and representing transcription activation and DNAbinding domains can be assembled into hybrid transcriptional activators.These hybrid transcriptional activators can be used with theircorresponding DNA elements (i.e., those bound by the DNA-binding SDFs)to effect coordinated expression of desired genes (Schwarz et al., Mol.Cell. Biol., 12:266 (1992) and Martinez et al., Mol. Gen. Genet.,261:546 (1999)).

The SDFs of the invention can also be used in the two-hybrid geneticsystems to identify networks of protein-protein interactions (L.McAlister-Henn et al., Methods 19:330 (1999), J. C. Hu et al., Methods20:80 (2000), M. Golovkin et al., J. Biol. Chem. 274:36428 (1999), K.Ichimura et al., Biochem. Biophys. Res. Comm. 253:532 (1998)). The SDFsof the invention can also be used in various expression display methodsto identify important protein-DNA interactions (e.g. B. Luo et al., J.Mol. Biol. 266:479 (1997)).

Promoters

The SDFs provided in Table 2 or Table 2 of any of the priority patentapplications are also useful as structural or regulatory sequences in aconstruct for modulating the expression of the corresponding gene in aplant or other organism (e.g., a symbiotic bacterium). For example,promoter sequences associated with SDFs provided in Table 2 or Table 2of any of the priority patent applications can be useful in directingexpression of coding sequences either as constitutive promoters or todirect expression in particular cell types, tissues, or organs or inresponse to environmental stimuli.

With respect to the SDFs provided in Table 2 or Table 2 of any of thepriority patent applications, a promoter is likely to be a relativelysmall portion of a genomic DNA (gDNA) sequence located in the first 2000nucleotides upstream from an initial exon identified in a gDNA sequenceor initial “ATG” or methionine codon or translational start site in acorresponding cDNA sequence. Such promoters are more likely to be foundin the first 1000 nucleotides upstream of an initial ATG or methioninecodon or translational start site of a cDNA sequence corresponding to agDNA sequence. In particular, the promoter is usually located upstreamof the transcription start site. The fragments of a particular gDNAsequence that function as elements of a promoter in a plant cell willpreferably be found to hybridize to gDNA sequences of SDFs provided inTable 2 or Table 2 of any of the priority patent applications at mediumor high stringency, relevant to the length of the probe and its basecomposition.

Promoters are generally modular in nature. Promoters can consist of abasal promoter that functions as a site for assembly of a transcriptioncomplex comprising an RNA polymerase (e.g., RNA polymerase II). Atypical transcription complex will include additional factors such asTF_(II)B, TF_(II)D, and TF_(II)E. Of these, TF_(II)D appears to be theonly one to bind DNA directly. The promoter might also contain one ormore enhancers and/or suppressors that function as binding sites foradditional transcription factors that have the function of modulatingthe level of transcription with respect to tissue specificity and oftranscriptional responses to particular environmental or nutritionalfactors, and the like.

Short DNA sequences representing binding sites for proteins can beseparated from each other by intervening sequences of varying length.For example, within a particular functional module, protein bindingsites may be constituted by regions of 5 to 60, preferably 10 to 30,more preferably 10 to 20 nucleotides. Within such binding sites, thereare typically 2 to 6 nucleotides that specifically contact amino acidsof the nucleic acid binding protein. The protein binding sites areusually separated from each other by 10 to several hundred nucleotides,typically by 15 to 150 nucleotides, often by 20 to 50 nucleotides. DNAbinding sites in promoter elements often display dyad symmetry in theirsequence. Often elements binding several different proteins, and/or aplurality of sites that bind the same protein, will be combined in aregion of 50 to 1,000 basepairs.

Elements that have transcription regulatory function can be isolatedfrom their corresponding endogenous gene, or the desired sequence can besynthesized, and recombined in constructs to direct expression of acoding region of a gene in a desired tissue-specific, temporal-specific,or other desired manner of inducibility or suppression. Whenhybridizations are performed to identify or isolate elements of apromoter by hybridization to the long sequences presented in Table 2provided herein or Table 2 of any of the priority patent applications,conditions are adjusted to account for the above-described nature ofpromoters. For example short probes, constituting the element sought,are preferably used under low temperature and/or high salt conditions.When long probes, which might include several promoter elements, areused or when hybridizing to promoters across species, low to mediumstringency conditions are preferred.

If a nucleotide sequence of an SDF such as those provided in Table 2 ofany of the priority patent applications, or part of the SDF, functionsas a promoter or fragment of a promoter, then nucleotide substitutions,insertions, or deletions that do not substantially affect the binding ofrelevant DNA binding proteins would be considered equivalent to theexemplified nucleotide sequence. It is envisioned that there areinstances where it is desirable to decrease the binding of relevant DNAbinding proteins to silence or down-regulate a promoter, or converselyto increase the binding of relevant DNA binding proteins to enhance orup-regulate a promoter. In such instances, polynucleotides representingchanges to the nucleotide sequence of the DNA-protein contact region byinsertion of additional nucleotides, by changes to identity of relevantnucleotides, including use of chemically-modified bases, or by deletionof one or more nucleotides are considered encompassed by the presentinvention. In addition, fragments of the promoter sequences described inTable 2 of any of the priority patent applications and variants thereofcan be fused with other promoters or fragments to facilitatetranscription and/or transcription in specific type of cells or underspecific conditions.

Promoter function can be assayed by methods known in the art, preferablyby measuring activity of a reporter gene operatively linked to thesequence being tested for promoter function. Examples of reporter genesinclude those encoding luciferase, green fluorescent protein, GUS, neo,cat, and bar.

UTRs and Junctions

Polynucleotides comprising untranslated (UTR) sequences and intron/exonjunctions are also within the scope of the invention. UTR sequencesinclude introns and 5′ or 3′ untranslated regions (5′ UTRs or 3′ UTRs).Fragments of the sequences shown in Table 2 can comprise UTRs andintron/exon junctions.

These fragments of SDFs, especially UTRs, can have regulatory functionsrelated to, for example, translation rate and mRNA stability. Thus,these fragments of SDFs can be isolated for use as elements of geneconstructs for regulated production of polynucleotides encoding desiredpolypeptides.

Introns of genomic DNA segments might also have regulatory functions.Sometimes regulatory elements, especially transcription enhancer orsuppressor elements, are found within introns. Also, elements related tostability of heteronuclear RNA and efficiency of splicing and oftransport to the cytoplasm for translation can be found in intronelements. Thus, these segments can also find use as elements ofexpression vectors intended for use to transform plants.

Just as with promoters, UTR sequences and intron/exon junctions can varyfrom those shown in Table 2 provided herein or Table 2 of any of thepriority patent applications. Such changes from those sequencespreferably will not affect the regulatory activity of the UTRs orintron/exon junction sequences on expression, transcription, ortranslation unless selected to do so. However, in some instances, down-or up-regulation of such activity may be desired to modulate traits orphenotypic or in vitro activity.

Coding Sequences

Isolated polynucleotides of the invention can include coding sequencesthat encode polypeptides comprising an amino acid sequence encoded bysequences described in Table 1 or 2 or an amino acid sequence presentedin Table 1 or 2.

A nucleotide sequence encodes a polypeptide if a cell (or a cell free invitro system) expressing that nucleotide sequence produces a polypeptidehaving the recited amino acid sequence when the nucleotide sequence istranscribed and the primary transcript is subsequently processed andtranslated by a host cell (or a cell free in vitro system) harboring thenucleic acid. Thus, an isolated nucleic acid that encodes a particularamino acid sequence can be a genomic sequence comprising exons andintrons or a cDNA sequence that represents the product of splicingthereof. An isolated nucleic acid encoding an amino acid sequence alsoencompasses heteronuclear RNA, which contains sequences that are splicedout during expression, and mRNA, which lacks those sequences.

Coding sequences can be constructed using chemical synthesis techniquesor by isolating coding sequences or by modifying such synthesized orisolated coding sequences as described above.

In addition to coding sequences encoding the polypeptide sequences ofTable 1 or 2, which can be native to corn, Arabidopsis, soybean, rice,wheat, and other plants, the isolated polynucleotides can bepolynucleotides that encode variants, fragments, and fusions of thosenative proteins. Such polypeptides are described below.

In variant polynucleotides generally, the number of substitutions,deletions, or insertions is preferably less than 20%; more preferablyless than 15%; and even more preferably less than 10%, 5%, 3%, or 1% ofthe number of nucleotides comprising a particularly exemplifiedsequence. It is generally expected that non-degenerate nucleotidesequence changes that result in 1 to 10, more preferably 1 to 5, andmost preferably 1 to 3 amino acid insertions, deletions, orsubstitutions will not greatly affect the function of an encodedpolypeptide. The most preferred embodiments are those wherein 1 to 20,preferably 1 to 10, most preferably 1 to 5 nucleotides are added to, ordeleted from and/or substituted in the sequences disclosed in Table 1 or2, or polynucleotides that encode polypeptides disclosed in Table 1 or2, or fragments thereof.

Insertions or deletions in polynucleotides intended to be used forencoding a polypeptide preferably preserve the reading frame. Thisconsideration is not so important in instances when the polynucleotideis intended to be used as a hybridization probe.

Native Polypeptides and Proteins

Polypeptides within the scope of the invention include both nativeproteins as well as variants, fragments, and fusions thereof.Polypeptides of the invention are those encoded by any of the sixreading frames of sequences shown in Table 1 or 2, preferably encoded bythe three frames reading in the 5′ to 3′ direction of the sequences asshown.

Native polypeptides include the proteins encoded by the sequences shownin Table 1 or 2. Such native polypeptides include those encoded byallelic variants.

Polypeptide and protein variants will exhibit at least 75% sequenceidentity to those native polypeptides of Table 1 or 2. More preferably,the polypeptide variants will exhibit at least 85% sequence identity, atleast 90% sequence identity, or at least 95%, 96%, 97%, 98%, or 99%sequence identity. Fragments of polypeptide or fragments of polypeptideswill exhibit similar percentages of sequence identity to the relevantfragments of the native polypeptide. Fusions will exhibit a similarpercentage of sequence identity in that fragment of the fusionrepresented by the variant of the native peptide.

Polypeptide and protein variants of the invention can exhibit at least75% sequence identity to those motifs or consensus sequences providedherein. More preferably, the polypeptide variants can exhibit at least85% sequence identity; at least 90% sequence identity; or at least 95%,96%, 97%, 98%, or 99% sequence identity. Fragments of polypeptides canexhibit similar percentages of sequence identity to the relevantfragments of the native polypeptide. Fusions will exhibit a similarpercentage of sequence identity in that fragment of the fusionrepresented by the variant of the native peptide.

Furthermore, polypeptide variants will exhibit at least one of thefunctional properties of the native protein. Such properties include,without limitation, protein interaction, DNA interaction, biologicalactivity, immunological activity, receptor binding, signal transduction,transcription activity, growth factor activity, secondary structure,three-dimensional structure, etc. As to properties related to in vitroor in vivo activities, the variants preferably exhibit at least 60% ofthe activity of the native protein; more preferably at least 70%, evenmore preferably at least 80%, 85%, 90% or 95% of at least one activityof the native protein.

One type of variant of native polypeptides comprises amino acidsubstitutions, deletions, and/or insertions. Conservative substitutionsare preferred to maintain the function or activity of the polypeptide.

Within the scope of percentage of sequence identity described above, apolypeptide of the invention may have additional individual amino acidsor amino acid sequences inserted into the polypeptide in the middlethereof and/or at the N-terminal and/or C-terminal ends thereof.Likewise, some of the amino acids or amino acid sequences may be deletedfrom the polypeptide.

Antibodies

Isolated polypeptides can be utilized to produce antibodies.Polypeptides of the invention can generally be used, for example, asantigens for raising antibodies by known techniques. The resultingantibodies are useful as reagents for determining the distribution ofthe antigen protein within the tissues of a plant or within a cell of aplant. The antibodies are also useful for examining the production levelof proteins in various tissues, for example in a wild-type plant orfollowing genetic manipulation of a plant, by methods such as Westernblotting.

Antibodies of the present invention, both polyclonal and monoclonal, maybe prepared by conventional methods. In general, the polypeptides of theinvention are first used to immunize a suitable animal, such as a mouse,rat, rabbit, or goat. Rabbits and goats are preferred for thepreparation of polyclonal sera due to the volume of serum obtainable,and the availability of labeled anti-rabbit and anti-goat antibodies asdetection reagents. Immunization is generally performed by mixing oremulsifying the protein in saline, preferably in an adjuvant such asFreund's complete adjuvant, and injecting the mixture or emulsionparenterally (generally subcutaneously or intramuscularly). A dose of50-200 μg/injection is typically sufficient. Immunization is generallyboosted 2-6 weeks later with one or more injections of the protein insaline, preferably using Freund's incomplete adjuvant. One mayalternatively generate antibodies by in vitro immunization using methodsknown in the art, which for the purposes of this invention is consideredequivalent to in vivo immunization.

Polyclonal antisera is obtained by bleeding the immunized animal into aglass or plastic container, incubating the blood at 25° C. for one hour,followed by incubating the blood at 4° C. for 2-18 hours. The serum isrecovered by centrifugation (e.g., 1,000×g for 10 minutes). About 20-50mL per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the method of Kohler andMilstein (Nature, 256: 495 (1975)), or modification thereof. Typically,a mouse or rat is immunized as described above. However, rather thanbleeding the animal to extract serum, the spleen (and optionally severallarge lymph nodes) is removed and dissociated into single cells. Ifdesired, the spleen cells can be screened (after removal ofnonspecifically adherent cells) by applying a cell suspension to aplate, or well, coated with the protein antigen. B-cells producingmembrane-bound immunoglobulin specific for the antigen bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for the production of antibodies which bind specifically tothe immunizing antigen (and which do not bind to unrelated antigens).The selected monoclonal antibody-secreting hybridomas are then culturedeither in vitro (e.g., in tissue culture bottles or hollow fiberreactors), or in vivo (as ascites in mice).

Other methods for sustaining antibody-producing B-cell clones, such asby EBV transformation, are known.

If desired, the antibodies (whether polyclonal or monoclonal) may belabeled using conventional techniques. Suitable labels includefluorophores, chromophores, radioactive atoms (particularly ³²P and¹²⁵I), electron-dense reagents, enzymes, and ligands having specificbinding partners. Enzymes are typically detected by their activity. Forexample, horseradish peroxidase is usually detected by its ability toconvert 3,3′,5,5′-tetramethylbenzidine (TNB) to a blue pigment,quantifiable with a spectrophotometer.

Variants

A type of variant of the native polypeptides comprises amino acidsubstitutions. Conservative substitutions, described above, arepreferred to maintain the function or activity of the polypeptide. Suchsubstitutions include conservation of charge, polarity, hydrophobicity,size, etc. For example, one or more amino acid residues within thesequence can be substituted with another amino acid of similar polaritythat acts as a functional equivalent, for example providing a hydrogenbond in an enzymatic catalysis. Substitutes for an amino acid within anexemplified sequence are preferably made among the members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine, and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

Within the scope of percentage of sequence identity described above, apolypeptide of the invention may have additional individual amino acidsor amino acid sequences inserted into the polypeptide in the middlethereof and/or at the N-terminal and/or C-terminal ends thereof.Likewise, some of the amino acids or amino acid sequences may be deletedfrom the polypeptide. Amino acid substitutions may also be made in thesequences; conservative substitutions being preferred.

One preferred class of variants are those that comprise (1) the domainof an encoded polypeptide and/or (2) residues conserved between theencoded polypeptide and related polypeptides. For this class ofvariants, the encoded polypeptide sequence is changed by insertion,deletion, or substitution at positions flanking the domain and/orconserved residues.

Another class of variants includes those that comprise an encodedpolypeptide sequence that is changed in the domain or conserved residuesby a conservative substitution.

Yet another class of variants includes those that lack one of the invitro activities, or structural features of the encoded polypeptides.One example is polypeptides or proteins produced from genes comprisingdominant negative mutations. Such a variant may comprise an encodedpolypeptide sequence with non-conservative changes in a particulardomain or group of conserved residues.

Fragments

Fragments of particular interest are those that comprise a domainidentified for a polypeptide encoded by an MLS of the instant inventionand variants thereof. Also, fragments that comprise at least one regionof residues conserved between an MLS encoded polypeptide and its relatedpolypeptides are of interest. Fragments are sometimes useful aspolypeptides corresponding to genes comprising dominant negativemutations.

Fusions

Of interest are chimeras comprising (1) a fragment of the MLS encodedpolypeptide or variants thereof of interest and (2) a fragment of apolypeptide comprising the same domain. For example, an AP2 helixencoded by a MLS provided in Table 2 of any of the priority patentapplications can be fused to a second AP2 helix from ANT protein, whichcomprises two AP2 helices. The present invention also encompassesfusions of MLS encoded polypeptides, variants, or fragments thereoffused with related proteins or fragments thereof.

Definition of Domains

The polypeptides of the invention can possess identifying domains asindicated in Table 1. Domains are fingerprints or signatures that can beused to characterize protein families and/or motifs. Such fingerprintsor signatures can comprise conserved (1) primary sequence, (2) secondarystructure, and/or (3) three-dimensional conformation. Generally, eachdomain has been associated with either a family of proteins or a motif.Typically, these families and motifs have been correlated with specificin vitro and/or in vivo activities. Usually, the polypeptides withdesignated domain(s) can exhibit at least one activity that is exhibitedby any polypeptide that comprises the same domain(s).

Specific domains within the MLS-encoded polypeptides can be indicated inTable 1. In addition, the domains with the MLS-encoded-polypeptide canbe defined by the region that exhibits at least 70% sequence identitywith a consensus sequence. Protein domain descriptions can be obtainedfrom Prosite (Internet site: “expasy” dot “ch” slash “prosite” slash)(contains 1030 documentation entries that describe 1366 differentpatterns, rules and profiles/matrices), and Pfam (Internet site: “pfam”dot “wustl” dot “edu” slash “browse” dot “shtml”).

The particular sequences of identified SDFs can be provided in Table 2.One of ordinary skill in the art, having this data, can obtain clonedDNA fragments, synthetic DNA fragments or polypeptides constitutingdesired sequences by recombinant methodology known in the art.

Methods of Modulating Polypeptide Production

It is contemplated that polynucleotides provided herein can beincorporated into a host cell or in vitro system to modulate polypeptideproduction. For instance, the SDFs prepared as described herein can beused to prepare expression cassettes useful in a number of techniquesfor suppressing or enhancing expression.

An example are polynucleotides comprising sequences to be transcribed,such as coding sequences of the present invention, can be inserted intonucleic acid constructs to modulate polypeptide production. Typically,such sequences to be transcribed are heterologous to at least oneelement of the nucleic acid construct to generate a chimeric gene orconstruct.

Another example of useful polynucleotides are nucleic acid moleculescomprising regulatory sequences provided in Table 2 of any of thepriority patent applications. Chimeric genes or constructs can begenerated when the regulatory sequences are linked to heterologoussequences in a vector construct. Within the scope of invention are suchchimeric gene and/or constructs.

Also within the scope of the invention are nucleic acid molecules,whereof at least a part or fragment of these DNA molecules are presentedin Table 1 or 2 or polynucleotide encoding polypeptides presented inTable 1 or 2, and wherein the coding sequence is under the control ofits own promoter and/or its own regulatory elements. Such molecules areuseful for transforming the genome of a host cell or an organismregenerated from said host cell for modulating polypeptide production.

Additionally, a vector capable of producing the oligonucleotide can beinserted into the host cell to deliver the oligonucleotide.

More detailed description of components to be included in vectorconstructs are described both above and below.

Whether the chimeric vectors or native nucleic acids are utilized, suchpolynucleotides can be incorporated into a host cell to modulatepolypeptide production. Native genes and/or nucleic acid molecules canbe effective when exogenous to the host cell.

Methods of modulating polypeptide expression includes, withoutlimitation, suppression methods (such as antisense methods, ribozymemethods, co-suppression methods, methods involving inserting sequencesinto the gene to be modulated, and methods involving regulatory sequencemodulation) as well as methods for enhancing production (such as methodsinvolving inserting exogenous sequences and methods involving regulatorysequence modulation).

Suppression

Expression cassettes provided herein can be used to suppress expressionof endogenous genes which comprise the SDF sequence. Inhibitingexpression can be useful, for instance, to tailor the ripeningcharacteristics of a fruit (Oeller et al., Science, 254:437 (1991)) orto influence seed size (WO 98/07842) or to provoke cell ablation(Mariani et al., Nature, 357: 384-387 (1992)).

As described above, a number of methods can be used to inhibit geneexpression in plants, such as antisense, ribozyme, introduction ofexogenous genes into a host cell, insertion of a polynucleotide sequenceinto the coding sequence and/or the promoter of the endogenous gene ofinterest, and the like.

Antisense

An expression cassette as described above can be transformed into hostcell or plant to produce an antisense strand of RNA. For plant cells,antisense RNA inhibits gene expression by preventing the accumulation ofmRNA which encodes the enzyme of interest, see, e.g., Sheehy et al.,Proc. Nat. Acad. Sci. USA, 85:8805 (1988), and Hiatt et al., U.S. Pat.No. 4,801,540.

Co-Suppression

Another method of suppression is by introducing an exogenous copy of thegene to be suppressed. Introduction of expression cassettes in which anucleic acid is configured in the sense orientation with respect to thepromoter has been shown to prevent the accumulation of mRNA. A detaileddescription of this method is described above.

Insertion of Sequences into the Gene to be Modulated

Yet another means of suppressing gene expression is to insert apolynucleotide into the gene of interest to disrupt transcription ortranslation of the gene.

Homologous recombination could be used to target a polynucleotide insertto a gene using the Cre-Lox system (Vergunst et al., Nucleic Acids Res.,26:2729 (1998); Vergunst et al., Plant Mol. Biol., 38:393 (1998) andAlbert et al., Plant J., 7:649 (1995)).

In addition, random insertion of polynucleotides into a host cell genomecan also be used to disrupt the gene of interest (Azpiroz-Leehan et al.,Trends in Genetics, 13:152 (1997)). In this method, screening for clonesfrom a library containing random insertions is preferred for identifyingthose that have polynucleotides inserted into the gene of interest. Suchscreening can be performed using probes and/or primers described abovebased on sequences from Table 1 or 2 provided herein or Table 1 or 2 ofany of the priority patent applications, polynucleotides encodingpolypeptides set forth in Table 1 or 2 provided herein or Table 1 or 2of any of the priority patent applications, fragments thereof, andsubstantially similar sequence thereto. The screening can also beperformed by selecting clones or any transgenic plants having a desiredphenotype.

Genes Comprising Dominant-Negative Mutations

When suppression of production of the endogenous, native protein isdesired it is often helpful to express a gene comprising a dominantnegative mutation. Production of protein variants produced from genescomprising dominant negative mutations is a useful tool for research.Genes comprising dominant negative mutations can produce a variantpolypeptide which is capable of competing with the native polypeptide,but which does not produce the native result. Consequently, overexpression of genes comprising these mutations can titrate out anundesired activity of the native protein. For example, the product froma gene comprising a dominant negative mutation of a receptor can be usedto constitutively activate or suppress a signal transduction cascade,allowing examination of the phenotype and thus the trait(s) controlledby that receptor and pathway. Alternatively, the protein arising fromthe gene comprising a dominant-negative mutation can be an inactiveenzyme still capable of binding to the same substrate as the nativeprotein and therefore competes with such native protein.

Products from genes comprising dominant-negative mutations can also actupon the native protein itself to prevent activity. For example, thenative protein may be active only as a homo-multimer or as one subunitof a hetero-multimer. Incorporation of an inactive subunit into themultimer with native subunit(s) can inhibit activity.

Thus, gene function can be modulated in host cells of interest byinsertion into these cells vector constructs comprising a genecomprising a dominant-negative mutation.

Enhanced Expression

Enhanced expression of a gene of interest in a host cell can beaccomplished by either (1) insertion of an exogenous gene or (2)promoter modulation.

Insertion of an Exogenous Gene

Insertion of an expression construct encoding an exogenous gene canboost the number of gene copies expressed in a host cell.

Such expression constructs can comprise genes that either encode thenative protein that is of interest or that encode a variant thatexhibits enhanced activity as compared to the native protein. Such genesencoding proteins of interest can be constructed from the sequences fromTable 1 or 2 provided herein or Table 1 or 2 of any of the prioritypatent applications, polynucleotides encoding polypeptides set forth inTable 1 or 2 provided herein or Table 1 or 2 of any of the prioritypatent applications, fragments thereof, and substantially similarsequence thereto.

Such an exogenous gene can include either a constitutive promoterpermitting expression in any cell in a host organism or a promoter thatdirects transcription only in particular cells or times during a hostcell life cycle or in response to environmental stimuli.

Gene Constructs and Vector Construction

To use isolated SDFs of the present invention or a combination of themor parts and/or mutants and/or fusions of said SDFs in the abovetechniques, recombinant DNA vectors which comprise said SDFs and aresuitable for transformation of cells, such as plant cells, are usuallyprepared. The SDF construct can be made using standard recombinant DNAtechniques (Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nded., c. 1989 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) and can be introduced to the species of interest byAgrobacterium-mediated transformation or by other means oftransformation (e.g., particle gun bombardment) as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs, PACs, and vectorsof the sort described by

-   -   (a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA,        89:8794-8797 (1992); and Hamilton et al., Proc. Natl. Acad. Sci.        USA, 93:9975-9979 (1996);    -   (b) YAC: Burke et al., Science, 236:806-812 (1987);    -   (c) PAC: Sternberg et al., Proc. Natl. Acad. Sci. USA, January;        87(1):103-7 (1990);    -   (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl.        Acids. Res., 23:4850-4856 (1995);    -   (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et        al., J. Mol. Biol., 170:827-842 (1983) or Insertion vector,        e.g., Huynh et al., In: Glover N M (ed) DNA Cloning: A practical        Approach, Vol. 1 Oxford: IRL Press (1985);    -   (f) T-DNA gene fusion vectors: Walden et al., Mol. Cell. Biol.,        1:175-194 (1990); and    -   (g) Plasmid vectors: Sambrook et al., Molecular Cloning, a        Laboratory Manual, 2nd ed., c. 1989 by Cold Spring Harbor        Laboratory Press, Cold Spring Harbor, N.Y.

Typically, a vector will comprise the exogenous gene, which in turncomprises an SDF of the present invention to be introduced into thegenome of a host cell, and which gene may be an antisense construct, aribozyme construct, chimeraplast, or a coding sequence with any desiredtranscriptional and/or translational regulatory sequences, such aspromoters, UTRs, and 3′ end termination sequences. Vectors of theinvention can also include origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, introns, etc.

A DNA sequence coding for the desired polypeptide, for example a cDNAsequence encoding a full length protein, will preferably be combinedwith transcriptional and translational initiation regulatory sequenceswhich will direct the transcription of the sequence from the gene in theintended tissues of the transformed plant.

For example, for over-expression, a plant promoter fragment may beemployed that will direct transcription of the gene in all tissues of aregenerated plant. Alternatively, the plant promoter may directtranscription of an SDF of the invention in a specific tissue(tissue-specific promoters) or may be otherwise under more preciseenvironmental control (inducible promoters).

If proper polypeptide production is desired, a polyadenylation region atthe 3′-end of the coding region is typically included. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant genes, or from T-DNA.

The vector comprising the sequences from genes or SDF or the inventionmay comprise a marker gene that confers a selectable phenotype on plantcells. The vector can include promoter and coding sequence, forinstance. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or phosphinotricin.

Coding Sequences

Generally, the sequence in the transformation vector and to beintroduced into the genome of the host cell does not need to beabsolutely identical to an SDF of the present invention. Also, it is notnecessary for it to be full length, relative to either the primarytranscription product or fully processed mRNA. Furthermore, theintroduced sequence need not have the same intron or exon pattern as anative gene. Also, heterologous non-coding segments can be incorporatedinto the coding sequence without changing the desired amino acidsequence of the polypeptide to be produced.

Promoters

As explained above, introducing an exogenous SDF from the same speciesor an orthologous SDF from another species are useful to modulate theexpression of a native gene corresponding to that SDF of interest. Suchan SDF construct can be under the control of either a constitutivepromoter or a highly regulated inducible promoter (e.g., a copperinducible promoter). The promoter of interest can initially be eitherendogenous or heterologous to the species in question. Whenre-introduced into the genome of said species, such promoter becomesexogenous to said species. Over-expression of an SDF transgene can leadto co-suppression of the homologous endogenous sequence thereby creatingsome alterations in the phenotypes of the transformed species asdemonstrated by similar analysis of the chalcone synthase gene (Napoliet al., Plant Cell, 2:279 (1990) and van der Krol et al., Plant Cell,2:291 (1990)). If an SDF is found to encode a protein with desirablecharacteristics, its over-production can be controlled so that itsaccumulation can be manipulated in an organ- or tissue-specific mannerutilizing a promoter having such specificity.

Likewise, if the promoter of an SDF (or an SDF that includes a promoter)is found to be tissue-specific or developmentally regulated, such apromoter can be utilized to drive or facilitate the transcription of aspecific gene of interest (e.g., seed storage protein or root-specificprotein). Thus, the level of accumulation of a particular protein can bemanipulated or its spatial localization in an organ- or tissue-specificmanner can be altered.

Signal Peptides

SDFs containing signal peptides are indicated in Table 1 or 2 of any ofthe priority patent applications. In some cases, it may be desirable forthe protein encoded by an introduced exogenous or orthologous SDF to betargeted (1) to a particular organelle intracellular compartment, (2) tointeract with a particular molecule such as a membrane molecule, or (3)for secretion outside of the cell harboring the introduced SDF. Thiswill be accomplished using a signal peptide.

Signal peptides direct protein targeting, are involved inligand-receptor interactions, and act in cell to cell communication.Many proteins, especially soluble proteins, contain a signal peptidethat targets the protein to one of several different intracellularcompartments. In plants, these compartments include, but are not limitedto, the endoplasmic reticulum (ER), mitochondria, plastids (such aschloroplasts), the vacuole, the Golgi apparatus, protein storagevesicles (PSV) and, in general, membranes. Some signal peptide sequencesare conserved, such as the Asn-Pro-Ile-Arg amino acid motif found in theN-terminal propeptide signal that targets proteins to the vacuole(Marty, The Plant Cell, 11:587-599 (1999)). Other signal peptides do nothave a consensus sequence per se, but are largely composed ofhydrophobic amino acids, such as those signal peptides targetingproteins to the ER (Vitale and Denecke, The Plant Cell, 11:615-628(1999)). Still others do not appear to contain either a consensussequence or an identified common secondary sequence, for instance thechloroplast stromal targeting signal peptides (Keegstra and Cline, ThePlant Cell, 11:557-570 (1999)). Furthermore, some targeting peptides arebipartite, directing proteins first to an organelle and then to amembrane within the organelle (e.g., within the thylakoid lumen of thechloroplast; see Keegstra and Cline, The Plant Cell, 11:557-570 (1999)).In addition to the diversity in sequence and secondary structure,placement of the signal peptide is also varied. Proteins destined forthe vacuole, for example, have targeting signal peptides found at theN-terminus, at the C-terminus, and at a surface location in mature,folded proteins. Signal peptides also serve as ligands for somereceptors.

These characteristics of signal proteins can be used to more tightlycontrol the phenotypic expression of introduced SDFs. In particular,associating the appropriate signal sequence with a specific SDF canallow sequestering of the protein in specific organelles (plastids, asan example), secretion outside of the cell, targeting interaction withparticular receptors, etc. Hence, the inclusion of signal proteins inconstructs involving SDFs increases the range of manipulation of SDFphenotypic expression. The nucleotide sequence of the signal peptide canbe isolated from characterized genes using common molecular biologicaltechniques or can be synthesized in vitro.

In addition, the native signal peptide sequences, both amino acid andnucleotide, described in Table 1 or 2 provided herein or Table 1 or 2 ofany priority patent application can be used to modulate polypeptidetransport. Further variants of the native signal peptides described inTable 1 or 2 provided herein or Table 1 or 2 of any priority patentapplication are contemplated. Insertions, deletions, or substitutionscan be made. Such variants will retain at least one of the functions ofthe native signal peptide as well as exhibiting some degree of sequenceidentity to the native sequence.

Also, fragments of the signal peptides of the invention are useful andcan be fused with other signal peptides of interest to modulatetransport of a polypeptide.

Transformation Techniques

A wide range of techniques for inserting exogenous polynucleotides areknown for a number of host cells, including, without limitation,bacterial, yeast, mammalian, insect and plant cells.

Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g. Weising et al., Ann. Rev. Genet., 22:421 (1988), and Christou,Euphytica, v. 85, n.1-3:13-27, (1995).

DNA constructs of the invention may be introduced into the genome of thedesired plant host by a variety of conventional techniques. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using ballistic methods, such as DNA particlebombardment. Alternatively, the DNA constructs may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria (McCormac et al., Mol. Biotechnol., 8:199(1997); Hamilton, Gene, 200:107 (1997); Salomon et al., EMBO J., 3:141(1984); Herrera-Estrella et al., EMBO J. 2:987 (1983).

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described by Paszkowski etal. (EMBO J., 3:2717 (1984)). Electroporation techniques are describedby Fromm et al. (Proc. Natl. Acad. Sci. USA, 82:5824 (1985)). Ballistictransformation techniques are described by Klein et al. (Nature, 327:773(1987)). Agrobacterium tumefaciens-mediated transformation techniques,including disarming and use of binary or co-integrate vectors, are welldescribed in the scientific literature. See, for example, Hamilton,Gene, 200:107 (1997); Müller et al., Mol. Gen. Genet., 207:171 (1987);Komari et al., Plant J., 10:165 (1996); Venkateswarlu et al.,Biotechnology, 9:1103 (1991); Gleave, Plant Mol. Biol., 20:1203 (1992);Graves and Goldman, Plant Mot. Biol., 7:34 (1986); and Gould et al.,Plant Physiology, 95:426 (1991).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype and thus the desired phenotypesuch as seedlessness. Such regeneration techniques rely on manipulationof certain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker, which has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described elsewhere (Evans et al., ProtoplastsIsolation and Culture in “Handbook of Plant Cell Culture,” pp. 124-176,MacMillan Publishing Company, New York, 1983; and Binding, Regenerationof Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1988).Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyby Klee et al. (Ann. Rev. of Plant Phys., 38:467 (1987)). Regenerationof monocots (rice) is described by Hosoyama et al. (Biosci. Biotechnol.Biochem., 58:1500 (1994)) and by Ghosh et al. (J. Biotechnol., 32:1(1994)). The nucleic acids of the invention can be used to conferdesired traits on essentially any plant.

Thus, the invention has use over a broad range of plants, includingspecies from the genera Anacardium, Arachis, Asparagus, Atropa, Avena,Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

DEFINITIONS

“Percentage of sequence identity” as used herein is determined bycomparing two optimally aligned sequences over a comparison window,where the fragment of the polynucleotide or amino acid sequence in thecomparison window may comprise additions or deletions (e.g., gaps oroverhangs) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Optimal alignment ofsequences for comparison may be conducted by the local homologyalgorithm of Smith and Waterman, (Add. APL. Math., 2:482 (1981)), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.,48:443 1970), by the search for similarity method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis.), or by inspection. Giventhat two sequences have been identified for comparison, GAP and BESTFITare preferably employed to determine their optimal alignment. Typically,the default values of 5.00 for gap weight and 0.30 for gap weight lengthare used. The term “substantial sequence identity” betweenpolynucleotide or polypeptide sequences refers to polynucleotide orpolypeptide comprising a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90% and mostpreferably at least 95%, even more preferably, at least 96%, 97%, 98% or99% sequence identity compared to a reference sequence using theprograms.

“Stringency” as used herein is a function of probe length, probecomposition (G+C content), and salt concentration, organic solventconcentration, and temperature of hybridization or wash conditions.Stringency is typically compared by the parameter T_(m), which is thetemperature at which 50% of the complementary molecules in thehybridization are hybridized, in terms of a temperature differentialfrom T_(m). High stringency conditions are those providing a conditionof T_(m)-5° C. to T_(m)-10° C. Medium or moderate stringency conditionsare those providing T_(m)-20° C. to T_(m)-29° C. Low stringencyconditions are those providing a condition of T_(m)-40° C. to T_(m)-48°C. The relationship of hybridization conditions to T_(m) (in ° C.) isexpressed in the mathematical equation:

T _(m)=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)  (1)

where N is the length of the probe. This equation works well for probes14 to 70 nucleotides in length that are identical to the targetsequence. The equation below for T_(m) of DNA-DNA hybrids is useful forprobes in the range of 50 to greater than 500 nucleotides, and forconditions that include an organic solvent (formamide).

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L0.63(%formamide)  (2)

where L is the length of the probe in the hybrid. (P. Tijessen,“Hybridization with Nucleic Acid Probes” in Laboratory Techniques inBiochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993by Elsevier, Amsterdam). The T_(m) of equation (2) is affected by thenature of the hybrid; for DNA-RNA hybrids T_(m) is 10-15° C. higher thancalculated, for RNA-RNA hybrids T_(m) is 20-25° C. higher. Because theT_(m) decreases about 1° C. for each 1% decrease in homology when a longprobe is used (Bonner et al., J. Mol. Biol., 81:123 (1973)), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation (2) is derived assuming equilibrium and therefore,hybridizations according to the present invention are most preferablyperformed under conditions of probe excess and for sufficient time toachieve equilibrium. The time required to reach equilibrium can beshortened by inclusion of a hybridization accelerator such as dextransulfate or another high volume polymer in the hybridization buffer.

Stringency can be controlled during the hybridization reaction or afterhybridization has occurred by altering the salt and temperatureconditions of the wash solutions used. The formulas shown above areequally valid when used to compute the stringency of a wash solution.Preferred wash solution stringencies lie within the ranges stated above;high stringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m), and low stringency is 45-48° C. below T_(m).

What is claimed is:
 1. A nucleic acid comprising a first nucleic acid sequence and a second nucleic acid sequence heterologous to said first nucleic acid sequence, wherein said first nucleic acid sequence comprises a polypeptide coding sequence encoding a polypeptide having an amino acid sequence with at least 90 percent identity to the sequence set forth in SEQ ID NO:2. 